According to the Commission Regulation (EC) No. 1258/2011, the maximum allowed nitrate content of lettuce is defined within a broad range (2000–5000 mg NO3/kg), depending on harvest season and technology. This study focuses on the identification of the differences in nitrate accumulation between lettuce types and varieties, depending on production technology and on the investigation of the application of non-destructive FT-NIR spectroscopy for nitrate quantification, towards widely used UV–Vis spectroscopy.In the present study, combinations of seasons and technologies (spring × greenhouse, autumn × open field) were employed for the production of types (batavia, butterhead, lollo and oak leaf; both red and green colored); a total of 266 lettuce heads were analyzed. It was found that with standardized technology and conditions, autumn harvested green oak leaf lettuce types accumulated significantly less nitrate, than red oak or lollo leaf types. With spring harvested lettuces, batavia types generally accumulated generally more nitrates than butterhead types. Based on the linear discriminant analysis (LDA) of FT-NIR measurements the four distinct variety types diverge; the lollo type explicitly diverges from batavia and butterhead types. The LDA further revealed, that within lollo and oak leaf variety types, red and green leaved varieties diverge as well. A model was successfully built for the FT-NIR quantification of the nitrate content of lettuce samples (R2 = 0.95; RMSEE = 74.4 mg/kg fresh weight; Q2 = 0.90; RMSECV = 99.4 mg/kg fresh weight). The developed model is capable of the execution of a fast and non-invasive measurement; the method is suitable for the routine measurement of nitrate content in lettuce.